Many biotechnological applications require the use and manipulation of gene-sized polynucleotide fragments, including applications in metagenomics, metabolic engineering, and genetic analysis. Metagenomic studies have revealed a wealth of genes encoding novel biochemical pathways and biocatalysts that potentially could play important roles in industrial processes, such as the extraction of fuels from refractory petroleum deposits, the conversion of agricultural raw materials into bulk and specialty chemicals, the generation of fuels from renewable resources, the discovery and development of therapeutically useful products, and the like, e.g. Lorenz et al, Nature Reviews Microbiology, 3: 510-516 (2005); Handelsman, Microbiol. Mol. Biol. Rev., 68: 669-685 (2004); Van Hamme et al, Microbiol. Mol. Biol. Rev., 67: 503-549 (2003). It is expected that genes and genetic pathways discovered in metagenomics studies will provide an important source of raw materials for metabolic engineering, that is, the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of cells with the use of recombinant DNA technology, Bailey, Science, 252: 1668-1674 (1991); Lee et al, Curr. Opin. Biotech., 19: 556-563 (2008). In the field of genetic analysis, “padlock” probes and other large circular DNA probes provide effective detection of genetic variation and an approach to reducing genome complexity which could make personal genome sequencing feasible, e.g. Borodina et al, Anal. Biochem. 318: 309-313 (2003); Hardenbol et al, Nature Biotechnology, 21: 673-678 (2003); Nilsson et al, Nature Genetics, 16: 252-255 (1997); Porreca et al, Nature Methods, 4: 931-936 (2007); Turner et al, Nature Methods, 6: 315-316 (2009); Dahl et al, Nucleic Acids Research, 33: e71 (2005). However, all of these applications in metabolic engineering and genetic analysis depend on the availability of gene-sized DNA fragments that can be synthesized conveniently and inexpensively.
Phosphoramidite-based solid phase DNA synthesis has been a crucial technique for many, if not all, biotechnology applications involving nucleic acid manipulations. However, despite huge gains in efficiency over the years, its practical application is limited to the direct synthesis of polynucleotides having at most 100 to 200 bases, e.g. Hecker et al, Biotechniques, 24: 256-260 (1998). Because of this, many convergent or hierarchical synthesis approaches have been developed for assembling gene-sized fragments of DNA, i.e. fragments in the range of from one to several hundred bases to several thousand bases. In such approaches, sets of pre-synthesized pre-purified oligonucleotides specific for a desired sequence are custom synthesized and assembled into a gene-sized fragment using a variety of enzymatic techniques, e.g. Czar et al, Trends in Biotechnology, 27: 63-72 (2009); Tian et al, Nature, 432: 1050-1054 (2004); Xiong et al, FEMS Microbiol. Rev., 32: 522-540 (2008); Chen et al, J. Am. Chem. Soc., 116: 8799-8800 (1994). Unfortunately, none of these approaches provide a general solution to the increasing demand for inexpensive and conveniently manufactured gene-sized polynucleotides for applications in genetic engineering and analysis.
In view of the above, it would be useful to have available a technique for routine non-custom synthesis of large polynucleotide fragments for use in metabolic engineering and genetic analysis.